The lipid and protein phosphatase PTEN is a key tumor suppressor encoded by a single gene, but antibodies against PTEN recognize two distinct proteins, prompting Liang et al. to investigate whether there may be another isoform encoded by PTEN. Inspection of the 5′-untranslated region of the mRNA revealed several potential CUG translation initiation codons, and construction of cDNA expression plasmids and mutation analysis indicated that the construct with CUG513 produced two forms of PTEN, the well-characterized short form (~55 kD) PTEN and a longer form (~70 kD) that the authors designate PTENα. The CUG start site was weaker than the AUG start site, and the two forms were independently synthesized, in that PTEN was produced in greater abundance than PTENα, and mutation of the start site for one form did not affect production of the alternate form. Analysis of a tagged PTEN-knockin mouse confirmed that the alternate initiation site was functional in vivo. In cells expressing tagged versions of PTEN or PTENα, PTEN was predominantly cytosolic and nuclear, whereas PTENα primarily colocalized with mitochondria. Cell fractionation studies indicated that endogenous PTENα associated with the inner mitochondrial membrane and matrix fractions. Enhancing synthesis of PTENα using a drug that inhibits AUG initiation and promotes CUG initiation increased cytochrome c oxidase (COX) activity. Introduction of PTENα in Pten–/– mouse cells, which have reduced basal COX activity compared with Pten+/+ cells, fully rescued COX activity, whereas introduction of PTEN only partially rescued COX activity. PTENα abundance correlated with the COX activity of various tissues, with heart and skeletal muscle having the highest amounts of each. TALEN-mediated knockout of PTENα, which did not affect PTEN expression, resulted in cells with morphologically abnormal mitochondria, reduced mitochondrial membrane potential, decreased COX activity, and reduced ATP synthesis. Although only a minor proportion of PTEN was detected in the mitochondria, the amount of PTEN associated with mitochondria was reduced in the absence of PTENα. Coimmunoprecipitation and affinity purification studies indicated that PTENα and PTEN interacted. Reconstitution of Pten–/– mouse cells with various combinations of PTENα, PTEN, and the PTEN-induced kinase PINK1 showed that introduction of PTENα with either PTEN or PINK1 enhanced ATP production. Thus, the data suggested that PTENα targets to the mitochondria and recruits a small amount of PTEN, where together they stimulate cellular respiration and ATP production through PINK1 and COX.